[Conservative treatment of the pectus carinatum]. / Tratamiento conservador del pectus carinatum.

INTRODUCTION: Pectus carinatum (PC) is a deformity that involves the protrusion of the anterior chest wall. It is 10 times less frequent than pectus excavatum. It has a progressive growth and is more common with men. There are two different types, the lower or condrocorporal which is the most common one, and the upper or condromanubrial. Most of the time there are no cardio-respiratory symptoms. OBJECTIVE: We present our experience in the orthopedic treatment of the pectus carinatum. METHOD: Retrospective review of patients treated in our hospital from 2002 until 2009. Patients were treated with observation, aerobic exercises, postural change and/or compression braces. Literature review was performed of the treatment for this pathology. RESULTS: 18 patients have been diagnosed with PC, 16 were men and 2 women. All were treated in a nonoperative way. Only 11 of them used a compression brace. We missed two follow-ups and another has just yet begun to achieve proper results. All the rest have had excellent results with nonoperative treatment. None of them have had a surgical treatment. CONCLUSION: The PC is a disease that most often is a cosmetic problem, with no impact on a cardio-respiratory level. Classically it has been a surgical entity. In our experience we have found that the orthopedic method is an effective alternative, safe and with a significant reduction in morbidity. But we need the collaboration of the patient to accept and maintain continuity in the use of the prostheses.

Differential expression of ras protooncogenes during in vitro differentiation of human erythroleukemia cells.

We have compared the expression of the ras protooncogene family (H-, K-, and N-ras) in leukemia cell differentiation utilizing as a model K562 and HEL erythroleukemia cells treated either with 1-beta-arabinofuranosylcytosine or 12-O-tetradecanoylphorbol-13-acetate (TPA). 1-beta-D-Arabinofuranosylcytosine induced terminal erythroid differentiation of K562 cells, while TPA induced myeloid differentiation of K562 and HEL cells, resulting in myelomonocytic-like cells expressing macrophagic and megakaryocytic markers. H-ras mRNA levels showed a dramatic decrease in K562 cells subjected to erythroid and myelomonocytic differentiation. The same result was found at the protein level for p21H-ras. Expression of K-ras and N-ras in K562 cells also decreased with differentiation, although significant mRNA levels remained despite cessation of cell proliferation. The decrease in K-ras expression was greater for TPA-treated cells than for 1-beta-arabinofuranosylcytosine-treated cells. TPA-induced myelomonocytic differentiation in HEL cells also resulted in a dramatic down-regulation of H-ras mRNA levels. Thus, by using a leukemia cell line able to differentiate along two different lineages, our results reveal a lineage-specific modulation of ras gene family expression.

Inhibition of Friend cells proliferation by spi-1 antisense oligodeoxynucleotides.

The spi-1 proto-oncogene encodes the transcription factor PU.1 which is normally expressed in all hematopoietic cell lineages except in T cell lines. During the murine acute erythroleukemia induced by the Friend retrovirus, SFFV, spi-1 deregulation by insertional mutagenesis results in the overexpression of Spi-1/PU.1 in the malignant proerythroblastic cell. To assess the Spi-1 role in the proliferation and the differentiation arrest of the Friend tumor cells we inhibited spi-1 gene expression in two Friend cell lines by using antisense oligodeoxyribonucleotides. Proliferation and cloning efficiency of both cell lines were significantly inhibited by spi1 antisense. This antiproliferative effect was not related to an apparent maturation of erythroleukemic cells demonstrating that repression of spi-1 expression is not sufficient per se to restore the ability of the proerythroblastic cells to spontaneously differentiate in mature erythroblasts. These data suggest that the spi-1 gene would be involved in the Friend leukemic process by promoting the proerythroblast to proliferate.

Differential regulation of Max and role of c-Myc during erythroid and myelomonocytic differentiation of K562 cells.

The protein Max binds to c-Myc and the heterodimer c-Myc/Max seems to be the active form in vivo. While the expression of c-myc is extensively regulated, no major changes in max expression have been reported so far with respect to differentiation. We have studied the expression of c-Myc and Max during in vitro differentiation of the bipotent human myeloid leukemia K562 cell line. This cell model system allowed us to compare c-Myc and Max expression during differentiation along erythroid (induced by 1-beta-D-arabinofuranosyl-cytosine) and myelomonocytic lineages (induced by 12-0-tetradecanoylphorbol-13-acetate). We found that c-myc expression decreased as a result of both differentiating treatments. The expression level of max remained unchanged during myelomonocytic differentiation. In contrast, max mRNA and protein were dramatically down-regulated during erythroid differentiation of K562 cells, thus demonstrating that max gene is subjected to regulation during differentiation. We also studied the expression of the other two described members of the c-Myc network: mxi1 and mad. mxi1 expression increased during erythroid differentiation but was strongly down-regulated during myelomonocytic differentiation of K562. mad was constitutively expressed during K562 erythroid differentiation and slightly increased during induction of the myelomonocytic pathway. We have obtained K562 sublines stably transfected with a zinc-inducible c-myc gene. In these clones the overexpression of c-Myc did not interfere with TPA-induced myelomonocytic differentiation. In contrast, erythroid differentiation was significantly inhibited upon c-myc induction despite the down-regulation of endogenous max expression. These results suggest a differential role for c-Myc in the human myeloid cell differentiation depending on the cell lineage.

Max and inhibitory c-Myc mutants induce erythroid differentiation and resistance to apoptosis in human myeloid leukemia cells.

We have used the human leukemia cell line K562 as a model to study the role of c-myc in differentiation and apoptosis. We have generated stable transfectants of K562 constitutively expressing two c-Myc inhibitory mutants: D106-143, that carries a deletion in the transactivation domain of the protein, and In373, that carries an insertion in the DNA-interacting region. We show here that In373 is able to compete with c-Myc for Max binding and to inhibit the transformation activity of c-Myc. K562 cells can differentiate towards erythroid or myelomonocytic lineages. K562 transfected with c-myc mutants showed a higher expression of erythroid differentiation markers, without any detectable effects in the myelomonocytic differentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic Max overexpression resulted in an increased erythroid differentiation, thus reproducing the effects of c-myc inhibitory mutants. We also studied the role of c-myc mutants and max in apoptosis of K562 induced by okadaic acid, a protein phosphatases inhibitor. The expression of D106-143 and In373 c-myc mutants and the overexpression of max reduced the apoptosis mediated by okadaic acid. The common biochemical activity of D106-143 and In373 is to bind Max and hence to titrate out c-Myc to form non-functional Myc/Max dimers. Similarly, Max overexpression would decrease the relative levels of c-Myc/Max with respect to Max/Max. The results support a model where a threshold of functional c-Myc/Max is required to maintain K562 cells in an undifferentiated state and to undergo drug-mediated apoptosis.

H-, K- and N-Ras inhibit myeloid leukemia cell proliferation by a p21WAF1-dependent mechanism.

Mutated ras genes are frequently found in human cancer. However, it has been shown that oncogenic ras inhibits growth of primary cells, through pathways involving p53 and the cell cycle inhibitors p16INK4a and p19ARF. We have analysed the effect of the ectopic expression of the three mammalian ras genes on the proliferation of K562 leukemia cells, which are deficient for p53, p16INK4a, p15INK4b and p19ARF genes. We have found that high expression levels of both wild-type and oncogenic H-, K- and N-ras inhibit the clonogenic growth of K562 cells. Induction of H-rasV12 expression in K562 transfectants retards growth and this effect is accompanied with an increase of p21WAF1 mRNA and protein levels. Furthermore, p21WAF1 promoter is activated potently by oncogenic ras and less pronounced by wild-type ras. This induction is p53-independent since a p21WAF1 promoter devoid of the p53 responsive elements is still activated by Ras. Finally, inhibition of p21WAF1 expression by an antisense construct partially overcomes the growth inhibitory action of oncogenic H-ras. Altogether, these results indicate that the antiproliferative effect of ras in myeloid leukemia cells is associated to the induction of p21WAF1 expression and suggest the existence of p19ARF and p16INK4a-independent pathways for ras-mediated growth inhibition.

c-Myc antagonizes the effect of p53 on apoptosis and p21WAF1 transactivation in K562 leukemia cells.

c-myc protooncogene positively regulates cell proliferation and overexpression of c-myc is found in many solid tumors and leukemias. In the present study we used the K562 human myeloid leukemia cell line as a model to study the functional interaction between c-Myc and p53. Using two different methods, we generated K562 transfectant cell lines with conditional expression of either c-Myc or p53. The cells expressed the p53Vall35 mutant, which adopts a wild-type conformation at 32 degrees C, while c-Myc induction was achieved with a zinc-inducible expression vector. We found that p53 in wild-type conformation induces growth arrest and apoptosis of K562. Expression of c-Myc significantly attenuated apoptosis and impaired the transcriptional activity of p53 on p21WAF1, Bax and cytomegalovirus promoters. The impairment of p21WAF1 transactivation by c-Myc was confirmed by transfection of a c-Myc-estrogen receptor fusion protein and by induction of c-myc by zinc in transfected cells. Also, p53-mediated up-regulation of p21WAF1 mRNA protein were significantly reduced by c-Myc, while Bax levels were unaffected. Consistently, c-Myc increased cyclin-dependent kinase 2 activity in K562 cells expressing p53 in wild-type conformation. These results suggest that c-Myc overexpression may antagonize the pro-apoptotic function of p53, thus providing a molecular mechanism for the frequently observed deregulation of c-myc in human cancer.

Down-regulation of c-myc gene is not obligatory for growth inhibition and differentiation of human myeloid leukemia cells.

Suppression of c-myc expression is observed during induced differentiation of several myeloid cell lines and it has been attributed to the cell growth arrest that accompanies terminal differentiation. To dissect the role of c-Myc in the proliferation-differentiation switch we have studied c-myc expression in K562 cells exposed to several chemical agents. This model system allowed us to discriminate between the growth arrest and differentiation phenomena as well as the induction of differentiation along two different lineages (erythroid and myelomonocytic). Our results showed that c-myc expression did not significantly decrease when growth inhibition is reversible, either by treatment with a differentiating agent such as hydroxyurea (which induced erythroid differentiation) or by a non-differentiating agent such as interferon-alpha. In contrast, c-myc expression decreased when cells underwent terminal differentiation, either along the myelomonocytic (by 12-O-tetradecanoylphorbol-13-acetate) or erythroid (by 1-beta-D-arabinofuranosylcytosine) lineages. These results indicated that c-myc down-regulation is not obligatory for growth arrest and non-terminal differentiation of human myeloid cells. In contrast, c-myc down-regulation occurred in terminal differentiation, but induction of myelomonocytic differentiation resulted in a greater loss of c-myc mRNA than induction of erythroid differentiation.

Preliminary experience in external quality control of RT-PCR PML-RAR alpha detection in promyelocytic leukemia.

To standardize the results obtained in PML/RAR alpha RT-PCR detection by laboratories of hospitals involved in the Spanish Program for Treatment of Hematological Malignancies (PETHEMA) LPA-96, designed for the treatment of acute promyelocytic leukemia (APL), cDNA samples obtained by reverse transcription of RNA from bone marrow samples of patients with APL were sent to participating laboratories. During the first year of this external quality assessment trial nine samples were tested by a maximum of 12 laboratories. The control gene was satisfactorily amplified in 90% of the samples (62 of 69 samples), supporting the adequacy of the cDNA to be used as control sample. There was an 83% concordance between laboratories for PML/RAR alpha detection with similar results for the type of PML/RR alpha rearrangements. However, 17% disagreement still remained, attributable to low sensitivity or inadequacy of methods followed. The results stressed the need for implementation of an external quality assessment scheme to ensure the standardization of the results.

[Gastric heterotopia and cleft palate. Deformation sequence or syndromic association?]. / Heterotopia gástrica y fisura palatina. Secuencia deformativa o asociación sindrómica?

Gastric heterotopia is more frequently in small intestine, gall bladder, biliary tract, Meckel's diverticulum, colon and rectum, but it can be found in other locations. It is especially rare in oral cavity. We only find one case that concurs with cleft palate. We present the case of a neonate with gastric heterotopia and cleft palate in addition to other congenital malformations not related at first. We make a brief revision of literature, showing the pathogenesis of heterotopia and its possible association with the cleft palate.

Novel CTCF binding at a site in exon1A of BCL6 is associated with active histone marks and a transcriptionally active locus.

BCL6 is a zinc-finger transcriptional repressor, which is highly expressed in germinal centre B-cells and is essential for germinal centre formation and T-dependent antibody responses. Constitutive BCL6 expression is sufficient to produce lymphomas in mice. Deregulated expression of BCL6 due to chromosomal rearrangements, mutations of a negative autoregulatory site in the BCL6 promoter region and aberrant post-translational modifications have been detected in a number of human lymphomas. Tight lineage and temporal regulation of BCL6 is, therefore, required for normal immunity, and abnormal regulation occurs in lymphomas. CCCTC-binding factor (CTCF) is a multi-functional chromatin regulator, which has recently been shown to bind in a methylation-sensitive manner to sites within the BCL6 first intron. We demonstrate a novel CTCF-binding site in BCL6 exon1A within a potential CpG island, which is unmethylated both in cell lines and in primary lymphoma samples. CTCF binding, which was found in BCL6-expressing cell lines, correlated with the presence of histone variant H2A.Z and active histone marks, suggesting that CTCF induces chromatin modification at a transcriptionally active BCL6 locus. CTCF binding to exon1A was required to maintain BCL6 expression in germinal centre cells by avoiding BCL6-negative autoregulation. Silencing of CTCF in BCL6-expressing cells reduced BCL6 mRNA and protein expression, which is sufficient to induce B-cell terminal differentiation toward plasma cells. Moreover, lack of CTCF binding to exon1A shifts the BCL6 local chromatin from an active to a repressive state. This work demonstrates that, in contexts in which BCL6 is expressed, CTCF binding to BCL6 exon1A associates with epigenetic modifications indicative of transcriptionally open chromatin.

Differential expression and phosphorylation of CTCF, a c-myc transcriptional regulator, during differentiation of human myeloid cells.

CTCF is a transcriptional repressor of the c-myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down-regulated during terminal differentiation in most cell lines tested; (ii) CTCF down-regulation is retarded and less pronounced than that of c-myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post-transcriptional levels.

Induction of apolipoprotein E expression during erythroid differentiation of human K562 leukemia cells.

Apolipoprotein E (ApoE) is the only apolipoprotein that is expressed in extrahepatic tissues. ApoE expression was studied in leukemia K562 cells differentiated towards erythroid or myelomonocytic lineages. When K562 cells were differentiated into the erythroid lineage by addition either of 1-beta-D-arabinofuranosylcytosine or hydroxyurea, an increase in ApoE mRNA and protein was detected. A weaker ApoE induction was also observed during phorbol ester-induced myelomonocytic differentiation. Previous work has associated ApoE expression to monocytic differentiation. The findings reported here indicate that ApoE overexpression is not associated with a specific lineage in myeloid differentiation and that may play a role in erythroid differentiation.

Translocation (10;11)(p13;p15) in an infant acute myeloid leukemia with MLL gene rearrangement.

Molecular rearrangements of the MLL gene at the 11q23 region have been identified in most cases of infant leukemia, regardless of the phenotype. We present a case of acute myeloid leukemia which coexpressed myeloid and lymphoid markers in a 12-month-old girl. Karyotype analysis revealed the presence of a thus far unreported translocation t(10;11)(p13;p15). Although no 11q23 abnormalities were cytogenetically detectable, an MLL gene molecular rearrangement was found.

Bisphosphoglycerate mutase and pyruvate kinase activities during maturation of reticulocytes and ageing of erythrocytes.

An increase in bisphosphoglycerate mutase (BPGM) and a decrease in pyruvate kinase (PK), i.e. a decrease in PK/BPGM ratio, was observed in red cell populations from anemic rats containing 95% down to 3% reticulocytes in blood. Such a ratio has been used to study the fractionation of recticulocytes, according to their degree of maturation, after counter-current distribution of those cell populations in dextranpoly (ethylene glycol) two-phase systems. When applying this procedure to the fractionation according to age of erythrocytes from normal rats, the decrease of PK with cellular age was observed without a significant variation in BPGM activity.

Surgery of subglottic stenosis in neonates and children.

The excellent management of patients in the different Intensive Care Units has decreased mortality but, as a side effect, we have to treat an increasing number of patients with airway problems secondary to prolonged intubation. The clinical records of patients diagnosed of acquired or congenital subglottic stenosis (SE) between 1990 and 1995 were retrospectively reviewed. Types of treatment included conservative, endoscopic, and open surgery: anterior cricoid split (ACS), anterior laryngotracheoplasty (ALTP) and anteroposterior laryngotracheoplasty (APLTP). 46 patients had SE: 7 congenital and 39 acquired. According to Cotton's classification 13 had grade I, 16 grade II, 12 grade III and none grade IV. Eleven of twelve cases treated conservatively did well (92%); one out of six patients managed endoscopically required further surgery (7%); good results were obtained in 5 of 7 cases treated by ACS (71 %); 8 out of 9 patients treated by ALTP did well (89%) and 7 out of 8 managed by APLTP had good results (87.5%). One iatrogenic suture dehiscence required further surgery. There is no statistical difference in the complication rate between patients treated conservatively and those treated by open surgery, while the mean hospital stay was higher in the latter (p < 0.05). An appropriate surgical technique should be offered to those patients with SE who do not do well with conservative management, since these techniques have yielded good results with a low rate of complications. Long-term follow-up shows the absence of recurrence.

[Presurgical orthopedic treatment for cleft lip and palate]. / Uso de la ortopedia prequirúrgica en pacientes con fisura palatina: nuestra experiencia.

INTRODUCTION: A multidisciplinary approach with several specialits allows a complete treatment for Cleft Lip and Palate. We show our experience in presurgical orthopedic treatment in these patients, their advantages, their problems and the results. MATERIAL AND METHODS: Since 1999 presurgical orthopedy has been applied to 12 patients (3 bilateral cleft lip and palate and 9 unilateral cleft lip and palate). This approach was applied when there was a long distance between the alveolar segments. A palate mould and the location of Latham's appliance have been made in the operating room under general anesthesia. The patients were controlled by the orthodoncist and Latham's appliance was removed when cleft lip was closed. RESULTS: Latham's appliance was kept for 4-7 weeks with once a week controls until the distance between the maxillary segments was less than 1 mm; in bilateral cases of cleft lip and palate the premaxilla was moved between lateral segments. Then, lip closure and nasoplasty was made and, sometimes, an obturador was placed. CONCLUSIONS: Latham's appliance permit to achieve a perfect alignment of alveolar segments decreasing the soft tissues tension and facilitating the lip surgery, thus, a better aesthetic and functional results can be achieved. A more anatomic position of palate can be made and easier future orthopedic treatments are possible.

[Our experience in neonatal surgery in a 15-year period. Follow-up of 1,003 cases]. / Nuestra experiencia en cirugía neonatal a lo largo de 15 años. Seguimiento de 1.003 casos.

From 1982 to 1994, 1.003 cases of surgical newborns have been retrospectively studied. Frequency and epidemiology have been evaluated, so as associated malformations. Prenatal diagnosis was made in 72 cases (7.1%). The most frequent entity was intestinal atresia, with 122 cases (12.1%), followed by necrotizing enterocolitis in 80 cases (7.9%), nevertheless this last one was the most frequent finding in preterm newborns. Esophageal atresia was found in 57 cases (5.6%). Association with other malformations was found in 127 cases (12.6%), and 32 cases of them (3.1%) constituted congenital malformations syndromes. The number of surgical newborns has increased in the last years, mainly due to ambulatory surgery. Global mortality of operated patients was 62 cases (5.9%); the highest mortality index was for congenital diaphragmatic hernia (33.5%), followed by necrotizing enterocolitis (28%). Mortality rate has decreased in recent years, due to improving in management and treatment of these patients, and the consolidation of surgical newborn units. Future efforts should be aimed for decreasing in mortality and improving the standard of life of these patients.

[Treatment of post-traumatic pancreatic pseudocyst by percutaneous Huisman's drainage]. / Tratamiento del pseudoquiste pancreático postraumático mediante drenaje percutáneo de Huisman.

Pancreatic pseudocysts is a complication of acute posttraumatic pancreatitis. They usually cause recurrent abdominal pain, nausea, vomiting and elevation of serum amylase levels. A history of epigastric blunt trauma, the before mentioned clinical signs and echographic or scanning studies may lead to a certain diagnosis. Although most of them resolve spontaneously, some persist and active therapeutic measures are required. Surgical internal drainage has been the operative technique of choice in children. Nevertheless, treatment can be achieved by percutaneous aspiration or drainage of pancreatic recurrent collections. We present our experience in two children with posttraumatic pancreatic pseudocyst, treated successfully by means of a percutaneous transabdominal pig-tail catheter (Huisman catheter). The technique of catheter placement and clinical aspects are discussed.

[Rectocolonic plasty using mechanical stapler as a surgical solution in complications of megacolon]. / Rectocoloplastia con sutura mecánica como solución quirúrgica en las complicaciones del megacolon.

Hirschsprung's disease surgically treated with Duhamel's technique in which no mechanical suture has been used, usually presents, as main complication, cronic constipation, due to fecalomas in the rectal pouch. In our experience (30 cases plus four patients sent to our hospital for reintervention), this complication is not present when mechanical suture is introduced to the Duhamel's Technique. This allows us to assure that perineal rectocoloplasty, with auto-suture material is a precise optional treatment, with excellent results and allows the chance of not going through laparotomy in those cases that require reintervention.